Research activity in the Molecular Neuroscience Section, LNC, NINDS, DIR in FY 04 focused on the following objectives: 1) To investigate by functional genomics and single-cell gene expression analysis the molecular components, particularly transactivating factors, in the oxytocin (OT) and vasopressin (VP) producing magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), that regulate the cell-specific expression of OT & VP, and 2) to study mechanisms underlying the regulation of the circadian rhythm of VP gene expression in the suprachiasmatic nucleus (SCN), and tyrosine hydroxylase(TH) gene expression in the brainstem. OT and VP producing MCNs in the HNS are the only neuronal phenotypes present in the rat supraoptic nucleus (SON). Laser microdissection of the SON, extraction and T7-based amplification of its RNAs, and analysis of the resulting cDNAs by hybridization on a 35, 319 element DNA microarray, provided a detailed composite view of the gene expression profile of the MCNs. The genes expressed in the SON were compared to total hypothalamus. Of the 26,000 unique genes on the array, 1385 were found to be expressed in the SON at levels more than two times greater than in the hypothalamus as a whole. Of these, 123 were expressed more than 3.4-fold higher in SON versus hypothalamus. Most of these preferentially expressed genes were not previously known to be expressed in the MCNs. Quantitative and double-label in situ hybridization histochemistry was used selectively to confirm a number of these microarray observations and to evaluate the osmotic regulation and cell-specific expression of these genes.. Hypoosmolality, also known as hyponatremia, produces a dramatic inhibition of the VP and OT gene expression in the SON. We examined the effect of hypoosmolality on global gene expression in the OT and VP MCNs using laser microdissection and DNA microarrays. Genes expressed in the SON, were compared under three different experimental conditions; normosmotic (control), the dDAVP-treated, and hypoosmolar conditions for each gene on the array. Among the 4,959 genes that showed a statistically significant difference between the normosmolar control and the hypoosmolar condition, 41 preferentially expressed genes defined as (expression ratio >=3.4) were downregulated, and 83 were upregulated more than two-fold. These genes represented a wide variety of functions, and of these, 14 genes that were downregulated and 30 genes that were upregulated in the hypoosmolar compared to the control SONs, were transcription related factors. Quantitative in situ hybridization histochemistry was selectively used to confirm a number of these microarray observations and to further evaluate their osmotic regulation. Using single-cell gene expression analysis we found that paternally expressed gene 3 (Peg3), which is an imprinted gene expressed exclusively from the paternal allele and encodes a Kruppel-type zinc finger-containing protein that is involved in maternal behavior, is very robustly expressed in the VP-MCNs.. We also found a robust expression in the hypothalamus of an RNA in the antisense direction to the 3' untranslated region of the Peg3 gene, which we designated APeg3. The APeg3 mRNA is about 1kb in size, and is primarily expressed in VP-MCNs. The full-length sequence of APeg3, determined by 5' and 3' RACE, contains an open reading frame and predicts a protein of 93 amino acids. Both Peg3 and APeg3 gene expression in the VP-MCNs increases during systemic hyperosmolality in vivo, indicating that both of these genes are osmoregulated. Organotypic cultures of rat hypothalamus were used to study the effects of various neurotransmitters (Ionotropic excitatory amino acid agonists, NMDA; AMPA, kainate, the metabotropic glutamate agonist, DHPG, and VIP) and potassium depolarization on the induction of c-Fos expression and VP heteronuclear RNA (hnRNA) levels in suprachiasmatic nucleus (SCN) in the presence or absence of tetrodotoxin (TTX). Using TTX enabled us to determine whether these effects are direct or transsynaptic. Our results confirmed that transynaptic communication within the SCN is necessary to maintain normal level of VP transcription during daytime and that calcium channels, predominantly the L-type, are significantly involved in this process. We also showed that forskolin induced transcription of VP in the SCN predominantly involves the MAP-kinase pathway. Our results show that the patterns of c-Fos induction in the SCN in organotypic slices induced by various EAAs, potassium depolarization and neurotransmitters differ form the patterns of increased of VP transcription caused by the same agents, and that VIP is the critical neurotransmitter in the SCN governing VP gene expression.The regulation of gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, was studied in brainstem noradrenergic nuclei, locus coeruleus (LC), A2 and A1, in vitro, using 1) a novel slice-explant model, in which these brainstem nuclei maintained a high survival of the noradrenergic neurons, an organotypic topology and the co-expression of two identifying markers in addition to TH, i.e., norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2), 2)a quantitive analysis of TH transcription using a labeled intronic probe to measure TH heteronuclear RNA (hnRNA), and 3) the use of tetrototoxin in the media to eliminate spontaneous neural activity in these nuclei, thereby providing a basal state as the starting point for the study of TH transcription under various pharmacological perturbations. In the presence of TTX, the adenylcyclase stimulator, forskolin, produced a 155% increase in LC, a 130% increase in A1, and a 220% increase in A2 in TH hnRNA as compared to control nuclei. This effect of forskolin was abolished in the LC and A1 by the PKA inhibitor, H89 (5?M), but not by the MAP kinase pathway (MEK) inhibitor, PD98059 (75?M). In contrast, the robust increase in TH transcription produced by forskolin in A2 neurons, was completely inhibited by PD98059, and only partially inhibited by H89, showing that induced TH transcription is mediated by different kinase pathways in specific central noradrenergic neuronal subtypes.